Enhanced infrared ray absorbing/emitting nanoparticles and on-site diagnosis kit using same

ABSTRACT

Disclosed is a diagnostic kit for quickly diagnosing a target material with high sensitivity using nanoparticles that absorb infrared light and emit infrared light, in which the nanoparticles are maintained in particle size and have enhanced emission intensity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. section 371, of PCTInternational Application No.: PCT/KR2018/002616, filed on Mar. 6, 2018,which claims foreign priority to Korean Patent Application No.:KR10-2017-0086300, filed on Jul. 7, 2017, and Korean Patent ApplicationNo.: KR10-2018-0009062, filed on Jan. 25, 2018, in the KoreanIntellectual Property Office, both of which are hereby incorporated byreference in their entireties.

TECHNICAL FIELD

The present invention relates to a diagnostic kit for quickly diagnosinga target material with high sensitivity using nanoparticles that absorbinfrared light and emit infrared light, in which the nanoparticles aremaintained in particle size and have enhanced emission intensity.

BACKGROUND ART

An on-site immunoassay diagnostic kit (a lateral flow immunoassay kit(LFA)) is a suitable diagnostic platform for the user to directly detecta target material on site because of the high economic feasibility andconvenience thereof.

Although the diagnostic kit is based on an immunoassay technique and maythus be applied to all tests in which an antigen and an antibody arepresent, the conventional kit is problematic because the color change ofthe test line has to be visually judged, and also because thesensitivity is lowered due to signal interference depending on the kindof specimen. For example, like a pregnancy diagnostic kit, there is arestriction in that a biomarker such as a HCG hormone should be presentat a high concentration in a specimen, or in that the type of specimenis limited.

Commercially available on-site immunoassay diagnostic kits include kitsfor detecting a target material using color change of goldnanoparticles, a visible light fluorescence signal of quantum dots, asin the following Patent Literature, or chemical signal amplification.

PATENT LITERATURE

Korean Patent No. 10-1053473 (Registration Date: Jul. 27, 2011) “Steroidhormone detection kit and method using quantum dots”

However, the kit using the color change of the gold nanoparticles haslow sensitivity, and the detection result thereof is not uniform due tothe influence of the specimen. The kit using the visible lightfluorescence signal of quantum dots has high sensitivity by virtue ofthe high emission efficiency of quantum dots compared to the kit usingthe gold nanoparticles, but is problematic in that ultraviolet light forquantum dot emission generates autofluorescence of the specimen and thekit constituents (plastics, various pads) and the fluorescence ofvisible light is interfered with by the specimen, and still affectssensitivity. The method using chemical signal amplification is notsuitable for an on-site diagnostic kit because large analysis equipmentand skilled experts are needed.

DISCLOSURE Technical Problem

Accordingly, an objective of the present invention is to provide adiagnostic kit for the detection of a target material with highsensitivity, without any influence from a specimen, using nanoparticlesthat absorb infrared light and emit infrared light, in which thenanoparticles are maintained in particle size and have enhanced emissionintensity.

Technical Solution

In order to accomplish the above objective, the present invention isimplemented by the following embodiments.

According to an embodiment of the present invention, nanoparticles ofthe present invention are characterized in that the nanoparticles aredoped with a rare earth element and a heterogeneous dopant and absorbinfrared light and emit infrared light.

According to another embodiment of the present invention, nanoparticlesof the present invention are characterized in that the nanoparticles aredoped with a rare earth element and absorb infrared light and emitinfrared light, and the nanoparticles are further doped with aheterogeneous dopant to increase the distortion of a crystal structurein the nanoparticles so as to enable sensitive electron transfer.

Also, the nanoparticles of the present invention are characterized inthat the emission intensity of the nanoparticles may be controlled byadjusting the kind or concentration of the heterogeneous dopant.

Also, the nanoparticles of the present invention are characterized inthat the rare earth element includes at least one selected from thegroup consisting of Y, Er, Yb, Tm and Nd.

Also, the nanoparticles of the present invention are characterized inthat the rare earth element includes 50 mol % of Y, 48 mol % of Yb and 2mol % of Tm.

Also, the nanoparticles of the present invention are characterized inthat the heterogeneous dopant includes at least one selected from thegroup consisting of Ca, Si, Ni and Ti.

Also, the nanoparticles of the present invention are characterized inthat the wavelength of infrared light that is absorbed and thewavelength of infrared light that is emitted are different from eachother so that there is no interference between the infrared light thatis absorbed and the infrared light that is emitted.

Also, the nanoparticles of the present invention are characterized inthat they absorb infrared light having a wavelength of 960 to 980 nm andemit infrared light having a wavelength of 750 to 850 nm.

Also, the nanoparticles of the present invention are characterized inthat they include a core layer comprising particles doped with a rareearth element, and a shell layer surrounding the core layer to thusreduce surface defects so as to improve surface uniformity and furtherdoped with a heterogeneous dopant.

Also, the nanoparticles of the present invention are characterized inthat they further include a coating layer formed on the outer surface ofthe shell layer through coating with a monomer or a polymer to thusincrease the dispersibility of the nanoparticles in a fluid andfacilitate immobilization of a capture agent.

Also, the nanoparticles of the present invention are characterized inthat the core layer is provided in the form of nanoparticles by mixing1-octadecene, oleic acid and a rare earth element to afford a homogenoussolution, which is then mixed with methanol containing sodium hydroxideand ammonium fluoride, stirred, and allowed to react at a predeterminedtemperature for a predetermined period of time, and the shell layer isformed at a predetermined thickness on the core layer by mixing1-octadecene, oleic acid, a rare earth element and a heterogeneousdopant to afford a homogenous solution, which is then mixed withmethanol containing sodium hydroxide and ammonium fluoride and with thecore layer, stirred, and allowed to react at a predetermined temperaturefor a predetermined period of time.

According to still another embodiment of the present invention, acapture-agent/nanoparticle conjugate of the present invention ischaracterized in that it includes nanoparticles, which absorb infraredlight and emit infrared light, and a capture agent binding to thenanoparticles so as to specifically bind to a target material, in whichthe nanoparticles are the nanoparticles of claim 10, and the captureagent binds to the coating layer.

Also, the capture-agent/nanoparticle conjugate of the present inventionis characterized in that the capture agent includes an antibody or anaptamer.

According to yet another embodiment of the present invention, adiagnostic kit of the present invention, which reacts with a targetmaterial by unidirectionally moving a specimen containing the targetmaterial, is characterized in that it includes acapture-agent/nanoparticle conjugate, specifically binding to the targetmaterial and absorbing infrared light and emitting infrared light, and asecond capture agent specifically binding to the target material, inwhich the target material bound to the capture-agent/nanoparticleconjugate binds to the second capture agent during movement of thespecimen, and the capture-agent/nanoparticle conjugate is thecapture-agent/nanoparticle conjugate of claim 12.

Also, the diagnostic kit of the present invention is characterized inthat it further includes a third capture agent specifically binding tothe capture agent.

Also, the diagnostic kit of the present invention is characterized inthat the capture-agent/nanoparticle conjugate moves together with thespecimen, the second capture agent and the third capture agent areimmobilized on the diagnostic kit by a predetermined interval, and whenthe diagnostic kit is irradiated with infrared light, thecapture-agent/nanoparticle conjugate bound to the second capture agentand the capture-agent/nanoparticle conjugate bound to the third captureagent emit infrared light.

Also, the diagnostic kit of the present invention is characterized inthat the second capture agent is immobilized on the test line of thediagnostic kit, the third capture agent is immobilized on the controlline of the diagnostic kit, and the test line is located in front of thecontrol line.

According to still yet another embodiment of the present invention, adiagnostic device of the present invention is characterized in that itincludes a diagnostic kit and an infrared light reader configured toaccommodate the diagnostic kit and to apply infrared light to thediagnostic kit and measure infrared light emitted from the diagnostickit to thus provide imaged data to an external terminal, in which thediagnostic kit is the diagnostic kit of claim 16.

Also, the diagnostic device of the present invention is characterized inthat the infrared light reader includes a housing, constituting theouter shape of the infrared light reader, and a controller configured toapply infrared light to the diagnostic kit located in the housing andinserted through an insertion groove in the housing and to measureinfrared light emitted from the diagnostic kit to thus provide theimaged data to the external terminal.

Also, the diagnostic device of the present invention is characterized inthat the controller includes an interface unit for exchanginginformation with the terminal, a battery for supplying power necessaryfor the operation of the controller, an irradiation unit for applyinginfrared light to a membrane of the diagnostic kit located in thehousing, an optical unit for photographing infrared light emitted fromthe membrane after the infrared light is applied to the membrane by theirradiation unit, and an image-processing unit for digitizing andoutputting the photographed image output from the optical unit.

Advantageous Effects

According to the embodiments of the present invention, the followingeffects can be obtained.

In the present invention, a substance to be detected by the user can bequickly diagnosed on site from various specimens such as saliva, blood,stool, beverages, and soil. Further, in the present invention,nanoparticles that absorb infrared light and emit infrared light ratherthan visible light are used, and thus sample permeation becomes possibledue to the long wavelength and a background signal does not occur.Thereby, since there is no interference between light absorption andlight emission, the substance to be detected by the user can be detectedwith high sensitivity. Furthermore, in the present invention, a targetmaterial can be detected with higher sensitivity by further enhancingthe emission intensity of nanoparticles that absorb infrared light andemit infrared light.

Therefore, the present invention can be applied to pathogens such asBacillus anthracis and botulinum neurotoxin, animal viruses such asfoot-and-mouth disease and avian influenza, diseases such as cancer andcardiovascular diseases, or biomarkers for pregnancy diagnosis.

DESCRIPTION OF DRAWINGS

FIG. 1 schematically shows a diagnostic kit according to an embodimentof the present invention;

FIG. 2 is a perspective view showing a diagnostic device according toanother embodiment of the present invention;

FIG. 3 is a block diagram showing the detailed configuration of thecontroller of an infrared light reader of FIG. 2

FIG. 4 shows TEM images and an emission spectrum of nanoparticlesaccording to an embodiment of the present invention;

FIG. 5 shows TEM images of nanoparticles manufactured using aheterogeneous dopant in different amounts;

FIG. 6 shows the results of emission spectra of nanoparticlesmanufactured using a heterogeneous dopant in different amounts;

FIG. 7 shows the results of analysis of the crystal structure ofnanoparticles manufactured using a heterogeneous dopant in differentamounts;

FIG. 8 shows the results of elemental analysis of nanoparticlesaccording to an embodiment of the present invention;

FIG. 9 shows the results of infrared spectroscopy of nanoparticlesaccording to an embodiment of the present invention;

FIG. 10 shows the results of measurement of surface charge of acapture-agent/nanoparticle conjugate according to an embodiment of thepresent invention;

FIG. 11 shows camera images for evaluating infrared light emissioncapability of nanoparticles according to an embodiment of the presentinvention;

FIG. 12 shows the results of absorption spectra of conventional goldnanoparticles;

FIG. 13 shows the results of emission spectra of nanoparticles accordingto an embodiment of the present invention; and

FIG. 14 shows camera images of the analytical results of a specimenusing the diagnostic kit according to an embodiment of the presentinvention.

DESCRIPTION OF THE REFERENCE NUMERALS IN THE DRAWINGS

-   -   1: sample pad 2: conjugation pad 3: membrane    -   4: absorption pad 5: test line 6: control line    -   7: substrate 100: capture-agent/nanoparticle conjugate 200:        diagnostic kit    -   300: infrared light reader 400: terminal

BEST MODE

Hereinafter, a detailed description will be given of enhancednanoparticles absorbing and emitting infrared light and an on-sitediagnostic kit using the same according to the present invention, withreference to the appended drawings. Unless otherwise defined, all termsused herein have the same meanings as those commonly understood by oneof ordinary skill in the art to which the present invention belongs. Ifthe meaning of a term used herein conflicts with the general meaningthereof, the definition used herein shall prevail. In the followingdescription of the present invention, detailed descriptions of knownconstructions and functions incorporated herein will be omitted whenthey may make the gist of the present invention unclear. As used herein,when any part “includes” any element, it means that other elements arenot precluded but may be further included, unless otherwise mentioned.

According to an embodiment of the present invention, an on-sitediagnostic kit using enhanced nanoparticles absorbing and emittinginfrared light is described with reference to FIG. 1, and the diagnostickit 200 is configured to include a sample pad 1, a conjugation pad 2, amembrane 3, an absorption pad 4 and a substrate 7, in which the samplepad 1, the conjugation pad 2, the membrane 3 and the absorption pad 4are sequentially connected in a direction of movement of the specimen onthe substrate 7.

The sample pad 1 is a pad that absorbs a specimen and enables thediffusion flow of a target material to be analyzed. In the presentinvention, the specimen indicates a substance suspected of containingthe target material to be analyzed, and may be referred to as a sample,and the target material refers to a substance to be analyzed for theconcentration or presence thereof.

The conjugation pad 2 is a pad configured to include acapture-agent/nanoparticle conjugate 100 and to receive the specimenmoved from the sample pad 1 to thus allow the capture-agent/nanoparticleconjugate 100 and the target material contained in the specimen to bindto each other. The capture-agent/nanoparticle conjugate 100 that bindsto the target material, which is an analyte in the specimen, may beincluded in a dry state in the conjugation pad 2. When a liquid specimenis applied to the sample pad 1, the liquid specimen is allowed to wetthe dry sample pad 1 therewith, and then moves to the conjugation pad 2,whereby the target material contained in the specimen specifically bindsto the capture agent of the capture-agent/nanoparticle conjugate 100.

The capture-agent/nanoparticle conjugate 100 includes nanoparticlesdoped with a rare earth element and a heterogeneous dopant and absorbinginfrared light and emitting infrared light, and a capture agent bindingto the nanoparticles to thus specifically bind to the target material.The capture-agent/nanoparticle conjugate specifically binds to thetarget material and absorbs infrared light and emits infrared light,rather than visible light, that is, enables sample permeation because ofthe long wavelength thereof and generates no background signal, and thusthere is no interference between light absorption and light emission,whereby the target material to be detected by the user may be detectedwith high sensitivity. According to the present invention, the substanceto be detected by the user may be quickly diagnosed on site in variouskinds of specimens such as saliva, blood, stool, beverages and soil, andthe present invention may be applied to a variety of fields, includingpathogens such as Bacillus anthracis and botulinum neurotoxin, animalviruses such as foot-and-mouth disease and avian influenza, diseasessuch as cancer and cardiovascular diseases, or biomarkers for pregnancydiagnosis.

The capture-agent/nanoparticle conjugate 100, which absorbs infraredlight upon irradiation with infrared light and emits infrared light, ischaracterized in that the wavelength of infrared light that is absorbedand the wavelength of the infrared light that is emitted are differentfrom each other (for example, infrared light of a long wavelength isabsorbed and infrared light of a short wavelength is emitted).Preferably, infrared light having a wavelength of 960 to 980 nm isabsorbed and infrared light having a wavelength of 750 to 850 nm isemitted (when infrared light having a wavelength of 750 to 850 nm isemitted, permeability to a biomaterial such as tissue may increase, thuspreventing the influence of specimens such as blood, stool, etc.).Furthermore, infrared light exhibits high transmittance even if thespecimen is an opaque mixed solution, and may thus be applied to variouskinds of specimens such as blood, stool, saliva, beverages, and soil,and it is possible to improve the signal-to-noise ratio withoutgenerating autofluorescence. Therefore, the diagnostic kit includes thecapture-agent/nanoparticle conjugate described above, thereby solvingthe problem of low sensitivity of the existing on-site immunoassaydiagnostic kit and thus maximizing the sensitivity of the on-siteimmunoassay diagnostic kit while maintaining the convenience andeconomic feasibility thereof.

The nanoparticles may be doped with a rare earth element, therebyproviding upconverting nanoparticles that absorb long-wavelength lightenergy and emit short-wavelength light energy through pyrolysissynthesis. Also, the nanoparticles may be further doped with aheterogeneous dopant, whereby the distortion of the crystal structure inthe nanoparticles may be increased to some extent, thus enabling verysensitive electron transfer. Accordingly, the nanoparticles may befurther increased in emission intensity without any significant changein the size of the nanoparticles.

In an embodiment of the invention, the nanoparticles may include atleast one selected from the group consisting of fluorides, oxides,halides, oxysulfides, phosphates, and vanadates. For example, at leastone selected from the group consisting of NaYF₄, NaYbF₄, NaGdF₄, NaLaF₄,LaF₃, GdF₃, GdOF, La₂O₃, Lu₂O₃, Y₂O₃ and Y₂O₂S may be included. The rareearth element with which the nanoparticles are doped may include alanthanide element, and the wavelength ranges of light absorbed andemitted by the nanoparticles may be controlled by adjusting the kind andconcentration of the rare earth element contained in the nanoparticles.Also, it is possible to provide nanoparticles free of interference ofthe absorption and emission wavelength ranges of infrared light byadjusting the kind and concentration of the rare earth element. Examplesof the rare earth element for attaining the above effects may include atleast one selected from the group consisting of Y, Er, Yb, Tm and Nd.More particularly, the rare earth element may include 45 to 55 mol % ofY, 43 to 52 mol % of Yb and 1.5 to 3 mol % of Tm. The emission intensityof the nanoparticles may be controlled by adjusting the kind orconcentration of the heterogeneous dopant. Also, examples of theheterogeneous dopant with which the nanoparticles are further doped mayinclude at least one selected from the group consisting of Ca, Si, Niand Ti.

The nanoparticles doped with the rare earth element and theheterogeneous dopant may be manufactured through a doping processtypically known in the art of the present invention, for example, aprocess disclosed in Qian et al., Small, 5: 2285-2290, 2009; Li et al.,Advanced Materials, 20:4765-4769, 2008; Zhao et al., Nanoscale,5:944-952, 2013; Li et al., Nanotechnology, 19:345606, 2008, which areincorporated herein by reference in their entirety.

The capture agent is configured to specifically bind to the targetmaterial contained in the specimen, and may include, for example, anantibody, an aptamer, etc., and binding of the capture agent and thenanoparticles doped with the rare earth element and the heterogeneousdopant includes, but is not limited to, any bond selected from amongionic bonds, covalent bonds, metal bonds, coordination bonds, hydrogenbonds, and van der Waals bonds.

The nanoparticles include a core layer comprising particles doped with arare earth element, a shell layer surrounding the core layer to thusreduce surface defects so as to improve surface uniformity and furtherdoped with a heterogeneous dopant, and a coating layer formed by coatingthe outer surface of the shell layer with a monomer or a polymer to thusincrease the dispersibility of the nanoparticles in a fluid andfacilitate the immobilization of the capture agent, in which the captureagent binds to the coating layer. The nanoparticles have a core-shellstructure, thus reducing surface defects to increase surface uniformity,and increasing the extent of monodispersion to maximize infrared lightemission efficiency, and the shell layer thereof is additionally dopedwith the heterogeneous dopant, thereby further increasing the infraredlight emission intensity. As for the capture-agent/nanoparticleconjugate, the nanoparticles may be surface-treated with a monomer or apolymer, thereby increasing the dispersibility in a fluid in thespecimen, such as water, and facilitating the immobilization of theantibody.

For example, the core layer is provided in the form of nanoparticles ina manner in which 1-octadecene, oleic acid and a rare earth element aremixed to form a homogeneous solution, and the homogeneous solution ismixed with methanol containing sodium hydroxide and ammonium fluoride,stirred, and then allowed to react at a predetermined temperature for apredetermined period of time, and the shell layer is formed at apredetermined thickness on the core layer in a manner in which1-octadecene, oleic acid, a rare earth element and a heterogeneousdopant are mixed to form a homogeneous solution, and the homogeneoussolution is mixed with methanol containing sodium hydroxide and ammoniumfluoride and with the core layer, stirred, and then allowed to react ata predetermined temperature for a predetermined period of time.

The polymer for forming the coating layer may include at least oneselected from the group consisting of polyacrylic acid (PAA),polyallylamine (PAAM), 2-aminoethyl dihydrogen phosphate (AEP),polyethylene glycol diacid, polyethylene glycol maleimide acid, andpolyethylene glycol phosphate ester. The coating layer may be formedthrough any process typically known in the art, for example, ligandengineering, such as ligand exchange or oleic acid oxidation, ligandattraction, layer-by-layer assembly, surface treatment usingsilanization, surface polymerization, and the like. Alternatively,surface treatment may be performed using a process disclosed in PhotonUpconversion Nanomaterials, Fan Zhang, Springer, 2015, which isincorporated herein by reference in its entirety.

The membrane 3 includes a test line 5, on which a second capture agentreactive to the target material contained in the specimen isimmobilized, and a control line 6, on which a third capture agentreactive to the capture agent of the capture-agent/nanoparticleconjugate 100 is immobilized, the test line 5 being located closer tothe conjugation pad 2 than the control line 6. The second capture agentmay be configured to specifically bind to or react with the targetmaterial, and may include an antibody, an aptamer, etc., and the thirdcapture agent may be configured to specifically bind to or react withthe capture agent, and may include an antibody, an aptamer, etc.

The target material specifically bound to the capture agent of thecapture-agent/nanoparticle conjugate 100 in the conjugation pad 2 movesto the membrane 3, and a portion thereof binds to the second captureagent and is thus immobilized on the test line 5, and a portion thereofmay be immobilized on the control line 6 through reaction between thecapture agent of the capture-agent/nanoparticle conjugate 100 and thethird capture agent.

Since the second capture agent reactive to the target material containedin the specimen is immobilized on the test line 5, whether or not thespecimen contains the target material to be analyzed and theconcentration thereof may be analyzed through presence or absence ofinfrared light emission thereof and through emission intensitymeasurement.

Since the third capture agent reactive to the capture agent of thecapture-agent/nanoparticle conjugate 100 is immobilized on the controlline 6, whether or not the analysis is effective may be judged bydetermining whether the specimen has moved to a necessary positionthrough presence or absence of infrared light emission of the controlline 6 and whether the capture agent works.

The absorption pad 4 is a pad that absorbs a fluid in the specimenpassing through the membrane 3, and the absorption pad 4 may function asa pump that enables the specimen to continuously move from the samplepad 1 to the membrane 3 by absorbing the fluid contained in the specimenmoving from the sample pad 1 to the membrane 3. The specimen may bemoved from the sample pad 1 to the absorption pad 4 through aspecimen-developing solution if necessary, depending on the capacity ofthe specimen. The specimen-developing solution may be a solutionincluding at least one selected from the group consisting of, forexample, PBS (phosphate buffer saline), KCl, NaCl, Tween 20, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and NaN₃, but isnot limited thereto.

The sample pad 1, the conjugation pad 2, the membrane 3, and theabsorption pad 4 may include a solid capillary support, and the solidcapillary support may be used without limitation, so long as it is aporous polymer capable of functioning as a solid capillary carrier ofchemical components such as an antigen, an antibody, an aptamer or ahapten or is a natural, synthetic or synthetically modified naturalmaterial having a plurality of pores, and the shape thereof is notlimited. For example, the solid capillary support may include at leastone selected from the group consisting of cellulosic material, paper,cellulose acetate, nitrocellulose, polyethersulfone, polyethylene,nylon, polyvinylidene fluoride (PVDF), polyester, polypropylene, silica,vinyl chloride, vinyl chloride-propylene copolymer, vinyl chloride-vinylacetate copolymer, inactivated alumina, diatomaceous earth, MgSO₄,cotton, nylon, rayon, silica gel, agarose, dextran, gelatin andpolyacrylamide. More particularly, the membrane may include at least onepolymer selected from the group consisting of nitrocellulose,polyethersulfone, polyethylene, nylon, polyvinylidene fluoride,polyester and polypropylene. Also, in an embodiment of the invention,the solid capillary support may be provided in the form of a rod, aplate, a tube, a bead, or the like.

The substrate 7 may be used without limitation, so long as it is able tosupport and transport the sample pad 1, the conjugation pad 2, themembrane 3 and the absorption pad 4, and the substrate may beliquid-impermeable so that the fluid contained in the specimen does notleak through the substrate. The substrate 7 may include, for example,glass, polystyrene, polypropylene, polyester, polybutadiene, polyvinylchloride, polyamide, polycarbonate, epoxide, methacrylate, polymelamineand the like.

Another embodiment of the present invention pertains to nanoparticlesthat are doped with a rare earth element and a heterogeneous dopant andalso that absorb infrared light and emit infrared light, and stillanother embodiment of the present invention pertains to acapture-agent/nanoparticle conjugate that specifically binds to a targetmaterial and that also absorbs infrared light and emits infrared light,rather than visible light. The nanoparticles are the same as thenanoparticles described above, and the capture-agent/nanoparticleconjugate is the same as the capture-agent/nanoparticle conjugate 100described above, and thus a detailed description thereof will beomitted.

Yet another embodiment of the present invention pertains to a diagnosticdevice. With reference to FIGS. 1 to 3, the diagnostic device includesthe diagnostic kit 200 as described above and an infrared light reader300.

The infrared light reader 300 is configured to accommodate thediagnostic kit 200 and to apply infrared light to the diagnostic kit 200and measure infrared light emitted from the diagnostic kit 200 to thusprovide imaged data to an external terminal 400, and includes a housing310, a cable 320, a controller 330, etc.

The housing 310 constitutes the outer shape of the infrared light reader300, and the housing 310 has an insertion groove 311 into which thediagnostic kit 200 is inserted. The cable 320 connects the controller330 of the infrared light reader 300 to the terminal 400, and theterminal 400 displays information transmitted from the controller 330,and may include a smartphone, a laptop computer, a tablet computer, orthe like.

The controller 330 functions to apply infrared light to the diagnostickit 200, which is located in the housing 330 and is inserted through theinsertion groove 311 in the housing 310, and to measure infrared lightemitted from the diagnostic kit 200 so as to provide the imaged data tothe external terminal 400, and includes an interface unit 331 forexchanging information with the terminal 400, a battery 332 forsupplying power necessary for the operation of the controller 330, anirradiation unit 333 for applying infrared light to the membrane 3 ofthe diagnostic kit 200 located in the housing 310, an optical unit 334for photographing infrared light emitted from the membrane 200 afterirradiation of the membrane 3 with infrared light by means of theirradiation unit 333, an image-processing unit 335 for digitizing andoutputting the photographed image output from the optical unit 334, anda control unit 336 for controlling the overall operation of thecontroller 330. For example, the irradiation unit 333 plays a role inapplying infrared light at a wavelength of 980 nm, and the optical unit334 includes a visible-light-blocking filter and anultraviolet-light-blocking filter in order to photograph infrared light.The diagnostic kit 200 where the specimen is dispensed is inserted intothe insertion groove 311 in the infrared light reader 300 and theinfrared light reader 300 is connected to the terminal 400, whereby theantigen detection result in the diagnostic kit 200 can be confirmed onthe terminal 400.

Still yet another embodiment of the present invention pertains to amethod of diagnosing a target material using the aforementioneddiagnostic device.

The method of diagnosing the target material includes injecting aspecimen containing a target material into the sample pad of thediagnostic kit, applying infrared light to the membrane of thediagnostic kit with the infrared light reader after passing the injectedspecimen through a test line and a control line included in the membraneof the diagnostic kit, and photographing and imaging the infrared lightemitted from the membrane irradiated with the infrared light with theinfrared light reader.

Particularly, the injecting the specimen containing the target materialinto the sample pad of the diagnostic kit may include injecting aspecimen containing a target material into the sample pad of thediagnostic kit, allowing the target material contained in the specimento specifically bind to the capture agent of thecapture-agent/nanoparticle conjugate in the conjugation pad by movingthe specimen to the conjugation pad, immobilizing a portion of thetarget material on the test line 5 through binding to the second captureagent and a portion of the target material on the control line 6 throughreaction of the capture agent of the capture-agent/nanoparticleconjugate 100 and the third capture agent by moving the target materialbound to the capture-agent/nanoparticle conjugate to the membrane, andabsorbing the target material, which is not immobilized on the test lineor the control line of the membrane, to the absorption pad through themembrane.

The injecting the specimen containing the target material into thesample pad of the diagnostic kit may further include adding aspecimen-developing solution dropwise on the sample pad of thediagnostic kit after injection of the specimen into the sample pad,thereby facilitating movement of the specimen in the diagnostic kit.

The applying the infrared light to the membrane of the diagnostic kitmay be performed in a manner in which infrared light may be appliedafter the lapse of a predetermined period of time after injection of thespecimen containing the target material into the sample pad of thediagnostic kit. Here, the predetermined period of time indicates thetime required to pass the specimen containing the target materialthrough the test line and the control line included in the membrane ofthe diagnostic kit, and is not limited, but may fall in the range of,for example, about 5 min to 30 min, and particularly about 5 min to 20min.

A better understanding of the present invention will be given throughthe following examples, which are set forth to illustrate but are not tobe construed as limiting the scope of the present invention.

<Example 1> Preparation of Nanoparticles Absorbing and Emitting InfraredLight

(1) Formation of Core

1-octadecene, oleic acid, yttrium acetate hydrate, ytterbium acetatehydrate and thulium acetate hydrate were mixed (particularly, 7 mL of1-octadecene and 3 mL of oleic acid were mixed with 0.4 mmol oflanthanide (comprising 50 mol % of Y, 48 mol % of Yb and 2 mol % ofTm)), and were then heated at 150° C. to afford a homogeneous solution,which was then cooled to 50° C. 5 mL of methanol containing 1 mmol NaOHand 1.6 mmol NH₄F was added to the homogeneous solution and stirred for30 min, thus forming a mixed solution. In order to remove methanol, themixed solution was maintained at 100° C. for 10 min, and was thenmaintained in an argon gas atmosphere at 290° C. for 1 hr 30 min. Afternatural cooling of the mixed solution, the nanoparticles wereprecipitated in ethanol and washed three times with cyclohexane andethanol, thus obtaining nanoparticles (core).

(2) Formation of Shell (Formation of UCNPs)

1-octadecene, oleic acid, yttrium acetate hydrate and calcium acetatehydrate were mixed (particularly, 7 mL of 1-octadecene and 3 mL of oleicacid were mixed with 0.2 mmol of a dopant (comprising 85 mol % oflanthanide (Y) and 15 mol % of a heterogeneous dopant (Ca))), and werethen heated at 150° C. to afford a homogeneous solution, which was thencooled to 50° C. 5 mL of methanol containing 1 mmol NaOH and 1.6 mmolNH₄F, the homogeneous solution and the nanoparticles (core) prepared in(1) of Example 1 were mixed and stirred for 30 min, thus forming a mixedsolution. In order to remove methanol, the mixed solution was maintainedat 100° C. for 10 min, and was then maintained in an argon gasatmosphere at 290° C. for 1 hr 30 min. After natural cooling of themixed solution, the nanoparticles were precipitated in ethanol andwashed three times with cyclohexane and ethanol, thus obtainingnanoparticles (core/shell, UCNPs) having a core-shell structure.

<Example 2> Preparation of Capture Agent (Antibody)-NanoparticleConjugate

(1) Formation of Coating Layer

The nanoparticles (core/shell) were coated with a polymer using a ligandengineering process. The nanoparticles prepared in (2) of Example 1 weredispersed in 13.4 mL of tetrahydrofuran to give a nanoparticle solution,and 100 mg of dopamine hydrochloride dispersed in 600 μL of distilledwater was added to the nanoparticle solution, thus forming a solutionmixed with nanoparticles, which was then maintained in an argon gasatmosphere at 50° C. for 5 hr. After natural cooling of the solutionmixed with nanoparticles, addition with 16 μL of hydrochloric acid andthen washing two times with distilled water were performed, thusobtaining amine-group-containing nanoparticles (NH₂—UCNPs).

(2) Antibody Binding (Formation of Antibody-Nanoparticle Conjugate)

1 μL of a solution formed by mixing 2.1 mg of SATA(N-succinimidyl-S-acetyl-thioacetate), 61 μL of dimethyl sulfoxide and182 μL of 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid) was added with 25 μg of an anti-nucleoprotein antibody (anti-humanCRP antibody, first antibody) capturing the nucleoprotein of avianinfluenza virus (H5N6), allowed to react at room temperature for 30 min,further added with 12.5 μL of a 0.5 M hydroxylamine hydrochloridesolution, and allowed to react for 2 hr, after which the reactionresidue was removed using a 100 k filter tube, thereby obtaining athiolated antibody. 1.875 mg of the amine-group-containing nanoparticlesprepared in (1) of Example 2, 1 mL of distilled water and 12.5 μL of 1 MHEPES buffer solution were mixed to give a first solution, and 18.8 mgof sulfo-SMCC (sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate) was added to 100 μL of a10 mM HEPES buffer solution to give a second solution. The firstsolution was mixed with 1 μL of the second solution and allowed to reactfor 2 hr, after which the reaction residue was removed using a 100 kfilter tube, thus obtaining maleimidated nanoparticles. The thiolatedantibody and the maleimidated nanoparticles were added to a HEPES buffersolution, allowed to react at 4° C. for 24 hr and then centrifuged, thusobtaining antibody-immobilized nanoparticles (antibody-nanoparticleconjugate).

<Example 3> Manufacture of Diagnostic Kit Using Antibody-NanoparticleConjugate

(1) A sample pad was thoroughly wetted with a 10 mM HEPES buffersolution containing 0.3% (w/v) PVP (polyvinylpyrrolidone), completelydried, and then cut to a size of 4 mm×20 mm. An absorption pad wasdehydrated before use. A nitrocellulose membrane was laminated on aplastic card (a substrate) using a laminator, after which the secondantibody (anti-nucleoprotein having an epitope different from that ofthe first antibody), reactive to the antigen contained in the specimen,was dispensed on the test line T and the third antibody (anti-goatantibody) reactive to the first antibody immobilized on theantibody-nanoparticle conjugate was dispensed on the control line Cusing an automatic dispenser, followed by drying at room temperature for48 hr. A conjugation pad was thoroughly wetted with a 10 mM HEPES buffersolution containing 2.0% (w/v) BSA (Bovine Serum Albumin), 2.0% (w/v)Tween 20, 2.5% (w/v) sucrose and 0.3% (w/v) PVP, followed by drying inan oven, dispensing of the antibody-nanoparticle conjugate solutionprepared in Example 2 thereon, and complete drying in an oven beforeuse.

(2) As shown in FIG. 1, the sample pad, the conjugation pad, themembrane located on the substrate and the absorption pad were disposedin an overlapping manner, fixed, and placed in a plastic box, thusmanufacturing a diagnostic kit.

<Test Example 1> Evaluation of Size, Shape and Emission Spectrum ofNanoparticles

(1) The nanoparticles (core) prepared in (1) of Example 1 and thenanoparticles (core/shell) prepared in (2) of Example 1 were evaluatedwith a Talos F200X TEM having an acceleration voltage of 200 kV, and theemission spectra of respective nanoparticle-dispersed solutions weremeasured using an NIR spectrometer through irradiation with infraredlight at 980 nm. The results are shown in FIG. 4.

(2) As shown in FIG. 4, the nanoparticles (core, core/shell) werespherical overall in shape and had a diameter of tens of nm, and thecore and the core/shell had different emission intensities. This isbecause the formation of the shell on the core reduces the incidence ofsurface defects, thus increasing surface uniformity and enhancing lightemission by the heterogeneous dopant.

<Test Example 2> Evaluation of Size, Shape and Emission Spectrum ofNanoparticles Depending on the Amount of Heterogeneous Dopant

(1) UCNPs were prepared in the same manner as in Example 1, with theexception that the total mol number of the dopant in Example 1 wasmaintained and that the ratio of lanthanide (Y) and heterogeneous dopant(Ca) was adjusted. In FIGS. 5 to 8, 0% Ca designates the dopantcomprising 100 mol % of lanthanide (Y), 5% Ca designates the dopantcomprising 95 mol % of lanthanide (Y) and 5 mol % of the heterogeneousdopant (Ca), 10% Ca designates the dopant comprising 90 mol % oflanthanide (Y) and 10 mol % of the heterogeneous dopant (Ca), 15% Cadesignates the dopant comprising 85 mol % of lanthanide (Y) and 15 mol %of the heterogeneous dopant (Ca), and 20% Ca designates the dopantcomprising 80 mol % of lanthanide (Y) and 20 mol % of the heterogeneousdopant (Ca).

(2) The UCNPs prepared in (2) of Example 1 and the UCNPs prepared in (1)of Test Example 2 were evaluated with a Talos F200X TEM having anacceleration voltage of 200 kV. The results are shown in FIG. 5. Theemission spectra of respective nanoparticle-dispersed solutions weremeasured using an NIR spectrometer through irradiation with infraredlight at 980 nm. The results are shown in FIG. 6.

(3) As shown in FIG. 5, the UNCPs were spherical overall in shape andhad a diameter of tens of nm, indicating that the diameter thereof wasnot greatly changed even by the addition of the heterogeneous dopant.Furthermore, as seen in FIG. 6, it can be found that the concentrationof the heterogeneous dopant that was added had an influence on theemission intensity.

<Test Example 3> Analysis of Crystal Structure of Nanoparticles andElemental Analysis Thereof

(1) Each of the solutions in which the nanoparticles prepared in (1) ofExample 1, (2) of Example 1 and (1) of Test Example 2 were dispersed wasdispensed on a silicon wafer, and the crystal structure of thenanoparticles was analyzed using an XRD-7000. The results are shown inFIG. 7. The elemental analysis of the nanoparticles prepared in (2) ofExample 1 was performed using a Talos F200X TEM having an accelerationvoltage of 200 kV. The results are shown in FIG. 8. Particularly,elemental analysis was conducted through energy dispersive X-rayspectroscopy mapping in the state in which the photography mode waschanged to obtain a dark field image in TEM.

(2) As shown in FIG. 7(a), the crystal structures of UCNPs having core,and lanthanide (Y) and heterogeneous dopant (Ca) at different ratioswere similar to some extent, and as shown in FIG. 7(b), in which the 15°to 20° portion of 2 theta is enlarged in the image of FIG. 7(a), thepeak value was shifted gradually to the left in the (100) crystalorientation. Thus, it can be found that the concentration of theheterogeneous dopant that is added has some influence on the crystalstructure.

(3) As shown in FIG. 8, Na, Y, and F were distributed throughout thenanoparticles, and Yb and Tm were distributed in the core layer and Cawas distributed in the shell layer, indicating that UCNPs in which theshell layer includes the heterogeneous dopant were efficientlysynthesized.

<Test Example 4> Evaluation of Antibody Binding to Nanoparticles

(1) The UCNPs prepared in (2) of Example 1, the NH₂—UCNPs prepared in(1) of Example 2 and the antibody-immobilized nanoparticles (Ab-UCNPs)prepared in (2) of Example 2 were subjected to Fourier transforminfrared spectroscopy using an iS10 Fourier transform infraredspectrophotometer. The results are shown in FIG. 9. Also, the NH₂—UCNPsand the antibody-nanoparticle conjugate (Ab-UCNPs) prepared in (2) ofExample 2 were measured for surface charge (zeta potential) using aZetasizer (Zetasizer Nano ZS90, Malvern). The results are shown in FIG.10.

(2) As shown in FIG. 9, UNCPs had peaks at 1457 and 1558 cm⁻¹corresponding to the asymmetric and symmetric vibrations of the COO—group, respectively, and transmission bands at 2853 and 2924 cm⁻¹,corresponding to the asymmetric and symmetric vibrations of —CH₂ in thealkyl chain of the oleic acid. NH₂—UCNPs had bands at 1635 and 3289 cm⁻¹corresponding to the C—N and N—H vibrations of the amine group,respectively, and Ab-UCNPs had peaks at 1540 and 1653 cm⁻¹ correspondingto the amide bond, from which the antibody was confirmed to bind to thenanoparticles.

(3) As shown in FIG. 10, the zeta potential of NH₂-UCNPs and Ab-UCNPschanged from 38.3 mV to 8.06 mV, and immobilization of the antibody onthe nanoparticles was confirmed.

<Test Example 5> Evaluation of Infrared Light Emission Capability ofNanoparticles

(1) Gold nanoparticles (GNPs) were dispersed in a buffer solution and aduck-stool-containing buffer solution and photographed using a normalcamera. The results are shown in FIG. 11(a). The nanoparticles (UCNPs)prepared in (2) of Example 1 were dispersed in a buffer solution and aduck-stool-containing buffer solution, photographed using a normalcamera, irradiated with infrared light at a wavelength of 980 nm, andphotographed using an infrared light camera for photographing aninfrared light image at 800 nm. The results are shown in FIG. 11(b).Moreover, the duck-stool-containing buffer solution (stool), thegold-nanoparticle-dispersed buffer solution (GNPs in buffer) and thegold-nanoparticle-dispersed duck-stool-containing buffer solution (GNPsin stool) were measured for absorption spectra using a UV/VIS/NIRspectrometer. The results are shown in FIG. 12. Also, theduck-stool-containing buffer solution (stool), the UCNP-dispersed buffersolution (UCNPs in buffer) and the UCNP-dispersed duck-stool-containingbuffer solution (UCNPs in stool) were irradiated with infrared light ata wavelength of 980 nm and measured for emission spectra using a NIRspectrometer. The results are shown in FIG. 13. The gold nanoparticleswere obtained by adding 20 mL of a 1 mM HAuCl₄ solution with 2 mL of a1% trisodium citrate dihydrate solution, followed by reaction for 10 minand centrifugation.

(2) As shown in FIG. 11(a), when the gold nanoparticles were dispersedin the buffer solution (buffer), a red color was observed, but a redcolor was not observed when the gold nanoparticles were dispersed in theopaque stool-containing buffer solution (stool). As shown in FIG. 11(b),when the nanoparticles prepared in (2) of Example 1 were irradiated withinfrared light at a wavelength of 980 nm, infrared light at a wavelengthof about 800 nm was emitted upon dispersion both in the buffer solution(buffer) and in the stool-containing buffer solution (stool)(represented by ‘Laser on’ in the drawings).

(3) As shown in FIG. 12, when the gold nanoparticles were dispersed inthe opaque stool-containing buffer solution, an absorption peak at awavelength of 550 nm was not observed, but as shown in FIG. 13, infraredlight at a wavelength of 800 nm was emitted in both the UCNP-dispersedbuffer solution and the UCNP-dispersed opaque stool-containing buffersolution.

<Test Example 6> Analysis of Specimen Using Diagnostic Kit

(1) A specimen-developing solution (comprising a H5N6 antigen and atransparent buffer solution) having different concentrations ofnucleoprotein (C-reactive protein (CRP)) of avian influenza virus (H5N6)was added dropwise on the sample pad of the diagnostic kit of Example 3.After 20 min, infrared light at a wavelength of 980 nm was appliedthereto, and photography with an infrared light camera was performed.The results are shown in FIG. 14(a).

(2) The test was performed in the same manner as in (1) of Test Example5, with the exception that the stool-containing buffer solution was usedin lieu of the transparent buffer solution. The results are shown inFIG. 14(b), and the emission intensities of two lines C, T are shown inFIG. 14(c).

(3) As shown in FIG. 14, the viral detection limit was 10^(3.5)EID₅₀/mL, and the emission intensity of the test line was increased withan increase in the viral concentration, from which the target materialof interest can be confirmed to be easily detected using the diagnostickit. Moreover, regardless of whether the transparent buffer solution orthe stool-containing buffer solution (opaque) was used in thespecimen-developing solution, it was not difficult to confirm the viraldetection results on the test line, from which the virus can beconcluded to be stably detected on site using the above diagnostic kit.

Although the preferred embodiments of the present invention have beendisclosed for illustrative purposes, those skilled in the art willappreciate that various modifications, additions and substitutions arepossible, without departing from the scope and spirit of the inventionas disclosed in the accompanying claims.

The invention claimed is:
 1. Nanoparticles that are used on-siteimmunoassay diagnostic kits and move with the specimen and absorbinfrared light and emit infrared light, doped with a rare earth elementand a heterogeneous dopant and absorbing infrared light and emittinginfrared light, wherein the nanoparticles comprises a core layer dopedwith a rare earth element and a shell layer surrounds the core layer forincreasing a uniformity of the core layer by reducing surface defectsand further doped with a heterogeneous dopant, wherein the core layer isprovided in a form of nanoparticles by heating a solution containing therare earth element to form a homogeneous solution, and mixing a mixedsolution containing a sodium compound and a fluorine compound with thecooled homogeneous solution, and reacting by applying heat, wherein theshell layer is formed on the core layer to a predetermined thickness bymixing the nanoparticle form of the core layer and the heterogeneousdopants in the mixed solution and then reacting by applying heat,wherein the core layer is in a form of a hexagonal and the nanoparticles have a round shape morphology, wherein the rare earth elementincludes 50 mol % of Y, 48 mol % of Yb and 2 mol % of Tm.
 2. Thenanoparticles of claim 1, wherein emission intensity of thenanoparticles is controlled by adjusting a kind or concentration of theheterogeneous dopant.
 3. Nanoparticles that are used on-site immunoassaydiagnostic kits and move with the specimen and absorb infrared light andemit infrared light, doped with a rare earth element and a heterogeneousdopant and absorbing infrared light and emitting infrared light, whereinthe nanoparticles comprises a core layer doped with a rare earth elementand a shell layer surrounds the core layer for increasing a uniformityof the core layer by reducing surface defects and further doped with aheterogeneous dopant, wherein the core layer is provided in a form ofnanoparticles by heating a solution containing the rare earth element toform a homogeneous solution, and mixing a mixed solution containing asodium compound and a fluorine compound with the cooled homogeneoussolution, and reacting by applying heat, wherein the shell layer isformed on the core layer to a redetermined thickness by mixing thenanoparticle form of the core layer and the heterogeneous dopants in themixed solution and then reacting by applying heat, wherein the corelayer is in a form of a hexagonal and the nano particles have a roundshape morphology, wherein the heterogeneous dopant includes at least oneselected from the group consisting of Ca, Si, Ni and Ti.
 4. Thenanoparticles of claim 1, wherein the nanoparticles are configured suchthat a wavelength of infrared light that is absorbed and a wavelength ofinfrared light that is emitted are different from each other so thatthere is no interference between infrared light that is absorbed andinfrared light that is emitted.
 5. The nanoparticles of claim 1, whereinthe nanoparticles absorb infrared light having a wavelength of 960 to980 nm and emit infrared light having a wavelength of 750 to 850 nm. 6.The nanoparticles of claim 1, wherein the nanoparticles further includea coating layer formed on an outer surface of the shell layer throughcoating with a monomer or a polymer to increase dispersibility of thenanoparticles in a fluid and facilitate immobilization of a captureagent.
 7. The nanoparticles of claim 3, wherein emission intensity ofthe nanoparticles is controlled by adjusting a kind or concentration ofthe heterogeneous dopant.
 8. The nanoparticles of claim 3, wherein therare earth element includes at least one selected from the groupconsisting of Y, Er, Yb, Tm and Nd.
 9. The nanoparticles of claim 3,wherein the nanoparticles are configured such that a wavelength ofinfrared light that is absorbed and a wavelength of infrared light thatis emitted are different from each other so that there is nointerference between infrared light that is absorbed and infrared lightthat is emitted.
 10. The nanoparticles of claim 3, wherein thenanoparticles absorb infrared light having a wavelength of 960 to 980 nmand emit infrared light having a wavelength of 750 to 850 nm.
 11. Thenanoparticles of claim 3, wherein the nanoparticles further include acoating layer formed on an outer surface of the shell layer throughcoating with a monomer or a polymer to increase dispersibility of thenanoparticles in a fluid and facilitate immobilization of a captureagent.